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Detection of ALT Associated Promyelocytic Leukemia Nuclear Bodies (APBs) by Immunofluorescence-FISH (IF-FISH)
免疫荧光原位杂交法(IF-FISH)检测ALT相关的前髓细胞性白血病核体(APB)   

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参见作者原研究论文

本实验方案简略版
Oncogene
Dec 2013

Abstract

The activation of functions that counteract the physiological shortening of telomeres in rapidly proliferating cell is prerequisite for the progression of cancer cells to full malignancy (Collado et al., 2007). In most human cancers, the length of telomere is maintained through up-regulation of telomerase whereas a telomerase-independent pathway, termed Alternative Lengthening of Telomeres (ALT) is active in about 10-15% of cancers (Johnson and Broccoli, 2007; Heaphy et al., 2011). One characteristic feature of ALT is the formation of ALT-associated Promyelocytic Leukemia nuclear bodies (APBs) (Lang et al., 2010; Yeager et al., 1999). APBs contain Promyelocytic Leukemia nuclear bodies (PML-NB) components such as PML, SP100 and SUMO, telomeric DNA and telomere associated proteins including the shelterin components TRF1, TRF2, POT1, TIN2, TPP1 and Rap1 (Yeager et al., 1999). In addition, APBs contain proteins involved in DNA repair. In particular, the presence of components of the homologous recombination machinery suggests that APBs may promote telomere elongation by facilitating the homologous recombination of telomeric templates (Nabetani et al., 2004; Stavropoulos et al., 2002). This is also supported by the requirement of the homologous recombination-associated MRN complex for APB formation (Wu et al., 2000). Furthermore, APBs are suggested to be active sites of ATM and ATR dependent DNA repair (Nabetani et al., 2004). Finally, the number of APBs increases in G2 phase of the cell cycle when recombination is mainly active (Grobelny et al., 2000). We have shown that infection of normal and malignant B lymphocytes with the human oncogenic herpesvirus Epstein-Barr virus (EBV) is associated with the induction of APBs and with numerous signs of chromosomal and genomic instability (Kamranvar et al., 2007; Kamranvar and Masucci, 2011; Kamranvar et al., 2013).

Here we describe a method for detection of APBs in human B-lymphocytes. The method can be applied with minor modifications to different cell types including adherent, suspension and primary cells.

Keywords: ALT (中高音), APB (APB), Telomere (端粒), PML (PML)

Materials and Reagents

  1. Cells (suspension cells)
  2. Formaldehyde (Merck KGaA, catalog number: K43634203 228 )
  3. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  4. BSA (Sigma-Aldrich, catalog number: A7906 )
  5. Blocking reagent (Roche Diagnostics, catalog number: 11096176001 )
  6. Maleic acid (Sigma-Aldrich, catalog number: M0375 )
  7. Deionized formamide (Merck KGaA, catalog number: K25761484 902 )
  8. Tris-HCl
  9. Green-fluorescent Alexa Fluor® 488 (Life Technologies, InvitrogenTM, catalog number: A-11034 ) or Red-fluorescent Alexa Fluor® 594 (Life Technologies, InvitrogenTM, catalog number: A-11005 )
  10. Ethanol (Kemetyl, catalog number: 200-578-6 )
  11. Cy3-TelG (PANAGENE, catalog number: F1006 )
  12. FITC-TelC (PANAGENE, catalog number: F1009 )
  13. DAPI (Vector Laboratories)
  14. Fixation buffer (see Recipes)
  15. Permeabilization buffer (see Recipes)
  16. IF blocking buffer (see Recipes)
  17. FISH blocking solution (see Recipes)
  18. Maleic acid buffer (see Recipes)
  19. PNA probes (see Recipes)
  20. Hybridizing solution (see Recipes)
  21. Washing solution (see Recipes)
  22. PML antibody (see Recipes)
  23. Secondary antibody (see Recipes)
  24. Dehydration solution (see Recipes)
  25. Mounting medium (see Recipes)

Equipment

  1. Microscope glass slide (76 x 26 mm)
  2. Coverslip (preferably circular 19 mm diameter)
  3. Cytospin or slide centrifuge (Cytospin3 SHANDON)
  4. Cytospin funnel with white filter card
  5. Metal holder
  6. Hydrophobic barrier pen (ImmEdge Pen, model: H-4000 )
  7. Hot plate preheated to a temperature 80 °C
  8. Coplin jar
  9. Moist chamber

Procedure

  1. Wash the suspension cells once with PBS and prepare a dilution of 0.5 x 106 cells per ml of PBS at RT.
  2. Pre-label the slides and make a small hydrophobic barrier circle (~1.3 cm diameter) with ImmEdge pen around the area where the cells will be placed and let it dry before cytospin. (Hydrophobic circle will be used to fill with the buffers for fixation, permeabilization and blocking buffer.)
  3. Prepare the slides mounted with the filter card and cytospin funnel in the metal holder as shown in the Figure 1.


    Figure 1. The figure illustrates the preparation of a slide for cytospin

  4. Load up to 100 µl of the prepared cells in each funnel and spin for 1 min at 600 rpm.
    Note: If the method will be applied to growing adherent cells on coverslip, the steps 1-4 can be skipped.
  5. Remove the slides from the holders and let the moisture of the cells dry out for a few sec before fixation with the fixation buffer for 10 min at RT.
  6. Wash the cells 2 x 5 min each with PBS.
  7. Permeabilize the cells with permeabilization buffer for 5 min at RT.
  8. Remove the permeabilization buffer and incubate the cells for one hour with the IF blocking buffer in moist chamber.
  9. Remove the IF blocking buffer and incubate the cells in moist chamber with the fresh IF blocking buffer containing PML antibody for 2 h at RT or overnight at 4 °C.
  10. Wash the cells gently 3 x 10 min each with PBS on a shaker. (Washing steps can be done in any glass or plastic microscope slide staining Jars.)
    Note: The slides should be protected from light in the next following steps.
  11. Incubate the cells in moist chamber for 30 min with the secondary antibody.
  12. Wash the cells gently 3 x 10 min each with PBS on a shaker.
  13. Re-fix the cells for 5 min with fixation buffer at RT.
  14. Wash the cells 3 x 10 min each in PBS.
  15. Dehydrate the cells in coplinjar containing ethanol series, consecutively 70%, 95%, 100% EtOH, for 5 min each.
  16. Aspirate the ethanol completely and let the slides dry for a couple of minutes in the dark.
  17. Place a drop of hybridizing solution containing the PNA probe on each slide and cover it with a coverslip.
  18. Denature the probes in hybridizing solution for 10 min at 80 °C by placing the slides on a pre-warmed hot plate.
  19. Incubate the slides in moist chamber for 2 h at RT.
  20. Wash the slides 2 x 15 min in washing solution.
  21. Wash the slides 3 x 5 min in PBS.
  22. Air-dry the slides at RT for 10 min.
  23. Mount the slides with one drop of Vectashield and cover with a coverslip.
  24. Seal the coverslip edges with clear nail polish and let it dry.
  25. Keep the slides at 4 °C in dark until microscopy.
  26. Visualize the APBs using a confocal microscope as fluorescence signals from PML bodies co-localized with telomere signals.
  27. A Cell containing two or more APBs can be scored as a ALT positive cell (Figure 2).

Representative data


Figure 2. Representative micrograph illustrating APBs positive U2OS cell in interphase. The arrows show three APB foci in which the green PML signal co-localizes with the red telomere signal. In some cell lines the PML antibody gives a weak diffuse fluorescent background that is easily distinguished from the bright fluorescence of the PML bodies.

Notes

  1. Rapidly proliferating cancer cells often have shorter telomeres and higher protein expression compared to slow growing primary cells. This may cause high background in immunofluorescence and weak telomere signals in FISH. To improve the method, the incubation time with the primary PML antibody should be reduced and less photobleaching telomeric probes should be used. Cy3-TelG usually last longer than FITC-TelC and Alexa Fluor® 488 produces less background than Alexa Fluor® 594. In cells with short telomeres and high background signals, Cy3-TelG and Alexa Fluor® 488 are recommended.

Recipes

  1. Fixation buffer
    3.7% formaldehyde in PBS (10-fold dilution of 37% commercial stock solution)
    Adjust the PH to 7.5
    The fixative should be freshly prepared for each experiment
  2. Permeabilization buffer
    0.5% Triton X-100 in PBS
  3. IF blocking buffer
    1% BSA and 0.1% Triton X-100 in PBS
  4. FISH blocking solution
    10% blocking reagent in maleic acid buffer: 10 g of blocking reagent should be dissolved in 100 ml of maleic acid buffer
    Adjust the pH to 7.5 with NaOH and store at 4 °C
  5. Maleic acid buffer
    100 mM maleic acid and 150 mM NaCl in dH2O
  6. PNA probes
    Cy3-TelG: Cy3-OO-TTAGGGTTAGGGTTAGGG 3’
    Stock concentration: 53 µM in dH2O
    FITC-TelC: FITC-OO-CCCTAACCCTAACCCTAA 3’
    Stock concentration: 111 µM in dH2O
    Store the stocks at 4 °C in the dark
  7. Hybridizing solution
    70% deionized formamide
    0.5% FISH blocking reagent (from 10% stock)
    10 mM Tris-HCl (pH 7.2)
    PNA telomeric probe: FITC-TelC (1:500-1,000 dilution from stock) or Cy3-TelG (1:3,000-5,000 dilution from stock)
    Prepare it freshly and protect it from light
  8. Washing solution
    70% formamide
    10 mM Tris-HCl (pH 7.2) in dH2O
  9. PML antibody
    Use 1:100-200 dilution in the IF blocking buffer
    Monoclonal or polyclonal PML Antibodies can be used
  10. Secondary antibody
    Green-fluorescent Alexa Fluor® 488 or red-fluorescent Alexa Fluor® 594 diluted 1:1,000 in IF blocking buffer
  11. Dehydration solution
    Ethanol series 70%, 95% and 100%
  12. Mounting medium
    Vectashield embedding medium containing DAPI

Acknowledgments

Some principles of the described protocol have been extracted from the protocols of Dr Titia de Lange`s Lab. This work was supported by grants awarded by the Swedish Cancer Society and the Karolinska Institute, Stockholm, Sweden.

References

  1. Collado, M., Blasco, M. A. and Serrano, M. (2007). Cellular senescence in cancer and aging. Cell 130(2): 223-233.
  2. Grobelny, J. V., Godwin, A. K. and Broccoli, D. (2000). ALT-associated PML bodies are present in viable cells and are enriched in cells in the G(2)/M phase of the cell cycle. J Cell Sci 113 Pt 24: 4577-4585.
  3. Heaphy, C. M., Subhawong, A. P., Hong, S. M., Goggins, M. G., Montgomery, E. A., Gabrielson, E., Netto, G. J., Epstein, J. I., Lotan, T. L., Westra, W. H., Shih Ie, M., Iacobuzio-Donahue, C. A., Maitra, A., Li, Q. K., Eberhart, C. G., Taube, J. M., Rakheja, D., Kurman, R. J., Wu, T. C., Roden, R. B., Argani, P., De Marzo, A. M., Terracciano, L., Torbenson, M. and Meeker, A. K. (2011). Prevalence of the alternative lengthening of telomeres telomere maintenance mechanism in human cancer subtypes. Am J Pathol 179(4): 1608-1615.
  4. Johnson, J. E. and Broccoli, D. (2007). Telomere maintenance in sarcomas. Curr Opin Oncol 19(4): 377-382.
  5. Kamranvar, S. A. and Masucci, M. G. (2011). The Epstein-Barr virus nuclear antigen-1 promotes telomere dysfunction via induction of oxidative stress. Leukemia 25(6): 1017-1025.
  6. Kamranvar, S. A., Chen, X. and Masucci, M. G. (2013). Telomere dysfunction and activation of alternative lengthening of telomeres in B-lymphocytes infected by Epstein-Barr virus. Oncogene 32(49): 5522-5530.
  7. Kamranvar, S. A., Gruhne, B., Szeles, A. and Masucci, M. G. (2007). Epstein-Barr virus promotes genomic instability in Burkitt's lymphoma. Oncogene 26(35): 5115-5123.
  8.  Lang, M., Jegou, T., Chung, I., Richter, K., Munch, S., Udvarhelyi, A., Cremer, C., Hemmerich, P., Engelhardt, J., Hell, S. W. and Rippe, K. (2010). Three-dimensional organization of promyelocytic leukemia nuclear bodies. J Cell Sci 123(Pt 3): 392-400.
  9. Nabetani, A., Yokoyama, O. and Ishikawa, F. (2004). Localization of hRad9, hHus1, hRad1, and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body. J Biol Chem 279(24): 25849-25857.
  10. Stavropoulos, D. J., Bradshaw, P. S., Li, X., Pasic, I., Truong, K., Ikura, M., Ungrin, M. and Meyn, M. S. (2002). The Bloom syndrome helicase BLM interacts with TRF2 in ALT cells and promotes telomeric DNA synthesis. Hum Mol Genet 11(25): 3135-3144.
  11. Wu, G., Lee, W.-H. and Chen, P.-L. (2000). NBS1 and TRF1 Colocalize at Promyelocytic Leukemia Bodies during Late S/G2 Phases in Immortalized Telomerase-negative Cells IMPLICATION OF NBS1 IN ALTERNATIVE LENGTHENING OF TELOMERES. J Biol Chem 275(39): 30618-30622.
  12. Yeager, T. R., Neumann, A. A., Englezou, A., Huschtscha, L. I., Noble, J. R. and Reddel, R. R. (1999). Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Res 59(17): 4175-4179.

简介

抵抗快速增殖细胞中端粒的生理学缩短的功能的激活是癌细胞进展为完全恶性肿瘤的前提条件(Collado等人,2007)。在大多数人类癌症中,通过端粒酶的上调维持端粒长度,而称为替代端粒长径(ALT)的端粒酶非依赖性通路在约10-15%的癌症中是有活性的(Johnson和Broccoli,2007; Heaphy et al。,2011)。 ALT的一个特征是ALT相关的早幼粒细胞白血病核体(APB)的形成(Lang等人,2010; Yeager等人,1999)。 APB含有早幼粒细胞白血病核体(PML-NB)组分如PML,SP100和SUMO,端粒DNA和端粒相关蛋白,包括shelterin组分TRF1,TRF2,POT1,TIN2,TPP1和Rap1(Yeager等,/em,1999)。此外,APB含有参与DNA修复的蛋白质。特别地,同源重组机器的组分的存在表明APB可以通过促进端粒模板的同源重组来促进端粒延长(Nabetani等人,2004; Stavropoulos等人,/em,2002)。这也得到了同源重组相关MRN复合物对APB形成的需求的支持(Wu等人,2000)。此外,APB被认为是ATM和ATR依赖性DNA修复的活性位点(Nabetani等人,2004)。最后,当重组是主要活性时,APB的数目在细胞周期的G2期增加(Grobelny等人,2000)。我们已经显示,用人致癌性疱疹病毒EB病毒(EBV)感染正常和恶性B淋巴细胞与APB的诱导和染色体和基因组不稳定性的许多迹象相关(Kamranvar等人, >,2007; Kamranvar和Masucci,2011; Kamranvar等人,2013)。
这里我们描述了用于检测人B淋巴细胞中的APB的方法。该方法可以应用于对包括粘附细胞,悬浮细胞和原代细胞在内的不同细胞类型的微小修改

关键字:中高音, APB, 端粒, PML

材料和试剂

  1. 细胞(悬浮细胞)
  2. 甲醛(Merck KGaA,目录号:K43634203208)
  3. Triton X-100(Sigma-Aldrich,目录号:T9284)
  4. BSA(Sigma-Aldrich,目录号:A7906)
  5. 封闭试剂(Roche Diagnostics,目录号:11096176001)
  6. 马来酸(Sigma-Aldrich,目录号:M0375)
  7. 去离子甲酰胺(Merck KGaA,目录号:K25761484902)
  8. Tris-HCl
  9. 绿色荧光Alexa Fluor 488(Life Technologies,Invitrogen TM,目录号:A-11034)或红色荧光Alexa Fluor 594 (Life Technologies,Invitrogen TM,目录号:A-11005)
  10. 乙醇(Kemetyl,目录号:200-578-6)
  11. Cy3-TelG(PANAGENE,目录号:F1006)
  12. FITC-TelC(PANAGENE,目录号:F1009)
  13. DAPI(Vector Laboratories)
  14. 固定缓冲区(参见配方)
  15. 渗透缓冲液(参见配方)
  16. IF阻塞缓冲区(参见配方)
  17. FISH阻断溶液(参见配方)
  18. 马来酸缓冲液(参见配方)
  19. PNA探针(参见配方)
  20. 杂交溶液(参见配方)
  21. 洗涤液(见配方)
  22. PML抗体(参见配方)
  23. 二抗(见配方)
  24. 脱水溶液(参见配方)
  25. 安装介质(参见配方)

设备

  1. 显微镜载玻片(76×26mm)
  2. 盖片(优选圆形19mm直径)
  3. Cytospin或载玻片离心机(Cytospin3 SHANDON)
  4. 带白色过滤卡的Cytospin漏斗
  5. 金属支架
  6. 疏水屏障笔(ImmEdge Pen,型号:H-4000)
  7. 热板预热至80°C
  8. Coplin瓶子
  9. 湿室

程序

  1. 用PBS洗涤悬浮细胞一次,并在室温下制备0.5×10 6个细胞/ml PBS的稀释液。
  2. 预先标记幻灯片,并用ImmEdge笔制作一个小的疏水屏障圈(直径约1.3厘米),放置细胞的区域,让它在细胞离心前干燥。 (疏水圈将用于填充用于固定,透化和阻断缓冲液的缓冲液。)
  3. 如图1所示,准备安装有过滤卡和细胞离心柱漏斗的载玻片在金属支架上

    图1.该图说明了cytospin
    的幻灯片的准备工作
  4. 在每个漏斗中加载至100μl制备的细胞,并以600rpm旋转1分钟。
    注意:如果该方法适用于盖玻片上生长的贴壁细胞,则可以跳过步骤1-4。
  5. 从固定器上取下载玻片,让细胞的水分干燥几秒钟,然后在室温下用固定缓冲液固定10分钟。
  6. 用PBS洗涤细胞2×5分钟
  7. 在室温下用透化缓冲液渗透细胞5分钟。
  8. 取出透化缓冲液,并在保湿室中用IF封闭缓冲液孵育细胞1小时
  9. 取出IF阻断缓冲液,并将细胞在潮湿的室中与新鲜的含有PML抗体的IF封闭缓冲液在室温孵育2小时或在4℃过夜。
  10. 在摇床上用PBS轻轻洗涤细胞3×10分钟。 (洗涤步骤可以在任何玻璃或塑料显微镜载玻片染色瓶中进行。) 注意:在接下来的步骤中,幻灯片应该避光。
  11. 孵育细胞在潮湿室与第二抗体30分钟
  12. 在摇床上用PBS轻轻洗涤细胞3次,每次10分钟
  13. 重新固定细胞在室温下固定缓冲液5分钟。
  14. 在PBS中每次洗涤细胞3×10分钟
  15. 将含有乙醇系列的连续70%,95%,100%EtOH的细胞脱水5分钟。
  16. 完全抽吸乙醇,让玻片在黑暗中干燥几分钟。
  17. 在每个载玻片上放置一滴含有PNA探针的杂交溶液,并用盖玻片覆盖
  18. 通过将载玻片放置在预热的热板上,在80℃下使杂交溶液中的探针变性10分钟。
  19. 在室温下孵育载玻片2小时。
  20. 在洗涤溶液中洗涤载玻片2×15分钟。
  21. 在PBS中洗涤载玻片3 x 5分钟。
  22. 在室温下将载玻片风干10分钟。
  23. 用一滴Vectashield装载幻灯片,盖上盖玻片。
  24. 用清澈的指甲油密封盖玻片边缘,让其干燥
  25. 保持幻灯片在4°C在黑暗中直到显微镜
  26. 使用共焦显微镜作为来自与端粒信号共定位的PML体的荧光信号可视化APB。
  27. 包含两个或更多个APB的细胞可以被评分为ALT阳性细胞(图2)

代表数据


图2.代表性的显微照片说明在间期中APB阳性的U2OS细胞。箭头显示三个APB病灶,其中绿色PML信号与红色端粒信号共定位。在一些细胞系中,PML抗体产生弱扩散荧光背景,其容易地与PML体的明亮荧光区分开。

笔记

  1. 与缓慢生长的原代细胞相比,快速增殖的癌细胞通常具有更短的端粒和更高的蛋白质表达。这可能导致高背景的免疫荧光和弱端粒信号FISH。为了改进该方法,应该减少与初级PML抗体的孵育时间,并且应该使用更少的漂白端粒探针。 Cy3-TelG通常比FITC-TelC更长,Alexa Fluor 488比Alexa Fluor 594产生更少的背景。在具有短端粒和高背景信号的细胞中,Cy3-建议使用TelG和Alexa Fluor ® 488。

食谱

  1. 固定缓冲区
    3.7%甲醛的PBS溶液(37%市售储备溶液的10倍稀释液) 将PH调整为7.5
    固定剂应该为每个实验准备好
  2. 渗透缓冲液
    0.5%Triton X-100的PBS溶液中
  3. IF阻塞缓冲区
    1%BSA和0.1%Triton X-100的PBS溶液中
  4. FISH阻断溶液
    在马来酸缓冲液中的10%封闭试剂:将10克封闭试剂溶解在100毫升马来酸缓冲液中 用NaOH调节pH至7.5,储存于4℃
  5. 马来酸缓冲液
    100mM马来酸和150mM NaCl,在dH 2 O中
  6. PNA探头
    Cy3-TelG:Cy3-OO-TTAGGGTTAGGGTTAGGG 3'
    储备浓度:53μM,在dH 2 O中
    FITC-TelC:FITC-OO-CCCTAACCCTAACCCTAA 3'
    储备浓度:111μM,在dH 2 O中
    将股票存储在4°C的黑暗
  7. 杂交溶液
    70%去离子甲酰胺 0.5%FISH封闭试剂(来自10%储液)
    10mM Tris-HCl(pH7.2) PNA端粒探针:FITC-TelC(来自库存的1:500-1,000稀释)或Cy3-TelG(来自库存的1:3,000-5,000稀释)
    准备新鲜,防止光照
  8. 洗涤溶液
    70%甲酰胺 10mM Tris-HCl(pH 7.2),在dH 2 O中
  9. PML抗体
    在IF阻断缓冲液
    中使用1:100-200稀释 可以使用单克隆或多克隆PML抗体
  10. 二抗
    绿色荧光Alexa Fluor 488或红色荧光Alexa Fluor ® 594在IF阻断缓冲液中以1:1000稀释。
  11. 脱水溶液
    乙醇系列70%,95%和100%
  12. 安装介质
    含有DAPI的Vectashield包埋介质

致谢

所述协议的一些原理已经从Titia de Lange的实验室的协议中提取。 这项工作得到瑞典癌症协会和瑞典斯德哥尔摩Karolinska研究所授予的资助。

参考文献

  1. Collado,M.,Blasco,M.A。和Serrano,M。(2007)。 癌症和衰老中的细胞衰老。细胞 130 2):223-233
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引用:Kamranvar, S. A. and Masucci, M. G. (2014). Detection of ALT Associated Promyelocytic Leukemia Nuclear Bodies (APBs) by Immunofluorescence-FISH (IF-FISH). Bio-protocol 4(23): e1303. DOI: 10.21769/BioProtoc.1303.
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